Oligonucleotides have important therapeutic applications in medicine. Oligonucleotides can be used to silence genes responsible for a particular disease, or change expression levels of genes in a manner that might be beneficial to an organism. Gene-silencing prevents formation of a protein product by inhibiting translation, affecting the stability of a particular RNA species, or by affecting the amount of transcription of a particular genetic locus. Importantly, gene-silencing agents are a promising alternative to traditional small, organic compounds that inhibit the function of the protein linked to the disease. siRNA, shRNA (small hairpin RNA), antisense RNA, and micro-RNAs are oligonucleotides that carry out gene silencing as described above.
RNA interference (RNAi) is a process in which RNAs called small-interfering RNAs or siRNAs inhibit expression of a gene that has an identical or nearly identical sequence (i.e. an intracellular RNA to which the inhibitory RNA is capable of hybridizing under physiological conditions). In many cases, inhibition is caused by degradation of the messenger RNA (mRNA) transcribed from the target gene. The mechanism and cellular machinery through which such RNAi-directed target RNA degradation occurs has been investigated using both genetic and biochemical approaches. In the case of dsRNA (represented either by transfected dsRNA, shRNA encoded by an introduced expression vector, or endogenous RNA that may be processed to become an active RNAi moiety), processing occurs in the cytoplasm of a cell; if necessary, the RNAi molecule (or its precursor) is first processed into RNA fragments 21 to 25 nucleotides long. These RNAi molecules can then be loaded into dicer complexes, where they direct cleavage of target RNA molecules.
The ability to specifically affect expression of a target gene by RNAi can be therapeutically beneficial as many diseases arise from the abnormal expression of a particular genetic locus, gene or group of genes. In many cases, therapeutic value may be derived by specifically inhibiting expression of the mutant form of a gene. In specific embodiments, RNAi can be used to inhibit or attenuate the expression of the deleterious gene and therefore alleviate symptoms of a disease or provide a treatment or cure. For example, genes contributing to a cancerous state, to viral replication, or to a dominant genetic disease such as myotonic dystrophy can be inhibited. Alternatively, indirect gene activation of pathways is also possible by, for example, down regulation of a suppressor gene. Inflammatory diseases such as arthritis can also be treated by inhibiting genes such as NF-κB, cyclooxygenase or cytokines. Examples of targeted organs include, for example, the liver, lung, pancreas, spleen, kidney, skin, brain, prostate, and heart.
Antisense methodology generally describes the complementary hybridization of synthetic nucleic acid sequences to mRNA or DNA such that the normal functions, such as protein synthesis, of these intracellular nucleic acids are disrupted. Hybridization is the sequence-specific hydrogen bonding via Watson-Crick base pairs of oligonucleotides to RNA or single-stranded DNA. Such base pairs are said to be complementary to one another. In one mechanism, hybridization arrest, the oligonucleotide inhibitor binds to the target polynucleotide and thus prevents the binding of essential proteins, most often ribosomes, to the polynucleotide by simple steric hindrance. Another means by which antisense oligonucleotides disrupt polynucleotide function is by hybridization to a target mRNA, followed by enzymatic cleavage of the targeted RNA by intracellular RNase H. Disruption of function may also occur through altered intracellular trafficking of a targeted RNA.
Micro-RNAs are a large group of small RNAs produced naturally in organisms, at least some of which regulate the expression of target genes. Micro-RNAs are formed from an approximately 70-nucleotide single-stranded hairpin precursor transcript by Dicer. In many instances, the micro-RNA is transcribed from a portion of the DNA sequence that previously had no known function. As such, these coding regions may in fact be considered genetic loci. Micro-RNAs are not translated into proteins but rather often bind to specific messenger RNAs and may affect translation of the bound RNA.
The intracellular delivery of various therapeutic compounds such as polynucleotides is compromised because the trafficking of many compounds into living cells is highly restricted by the complex membrane systems of the cell. Specific transporters allow the selective entry of nutrients or regulatory molecules, while excluding most exogenous molecules such as polynucleotides and proteins. Various strategies can be used to improve transport of polynucleotides into cells, including lipid carriers, biodegradable polymers, and various conjugate systems. The most well studied approaches for improving the transport of foreign polynucleotides into cells involve the use of viral vectors or cationic lipids and related cytofectins. Viral vectors can be used to transfer genes efficiently into some cell types, but they generally cannot be used to introduce chemically synthesized molecules into cells. An alternative approach is to use delivery formulations incorporating cationic lipids, which interact with polynucleotides through one end and lipids or membrane systems through another. Another approach to delivering biologically active compounds involves the use of conjugates. Conjugates are often selected based on the ability of certain molecules to be selectively transported into specific cells, for example via receptor-mediated endocytosis. By attaching an active compound to molecules that are actively transported across the cellular membranes, the effective transfer of that compound into cells or specific cellular organelles can be realized. In other cases, conjugates may be used to mediate incorporation of an active compound into a delivery vehicle. Alternatively, molecules able to penetrate cellular membranes without active transport mechanisms, for example, various lipophilic molecules, can be used to deliver compounds of interest.
Compositions and methods for improving the efficiency of systemic and local delivery of biologically active molecules, particularly polynucleotide therapeutics such as siRNA are needed. The present disclosure fulfills this need and provides additional advantages described herein.